Yeast Functional Assay Research Articles

Screen p53 mutations in hepatocellular carcinoma by FASAY: A novel splicing mutation

To establish a routine procedure for the detection of p53 mutations in hepatocellular carcinoma (HCC) surgical resections using the FASAY (functional analysis of separated alleles of p53 on yeast) procedure. p53 status was analyzed by FASAY and cDNA sequencing in 50 cases of HCC. After the extraction of RNA from the frozen tumor and corresponding normal tissues, reverse transcription RT-PCR was carried out using these samples. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies. Of the 50 specimens, 29 (58%) were positive (mutant) by FASAY. Sequencing analysis confirmed that all 29 FASAY positive tumors harbored mutations, and that no mutations were detectable in any FASAY negative tumors. In 29 p53 mutations, 22 mutations were point missense mutation, 5 were deletions and 2 were splicing mutations. A novel splice mutation on splice donor of intron 6 was reported, which could produce two different mRNAs, respectively using the nearest upstream and downstream recessive splice donor sites. FASAY is a sensitive method for detecting the various types of p53 mutations in HCC, suggesting that the yeast functional assay for the detection of p53 mutations may be essential for elucidating their clinical significance.

European Organization for Research and Treatment of Cancer – Breast Cancer Group (EORTC BCG)

This section provides current contact details and a summary of recent or ongoing clinical trials being coordinated by European Organization for Research and Treatment of Cancer – Breast Cancer Group (EORTC BCG). Clinical trials include: Postoperative adjuvant chemotherapy followed by adjuvant tamoxifen versus nil for node-negative and node-positive patients with operable breast cancer. EORTC Study No. 10901Randomized phase III study comparing short, intensive preoperative combination chemotherapy with similar therapy given postoperatively. EORTC Trial No. 10902Phase III randomized trial investigating the role of internal mammary and medial supraclavicular (IM-MS) lymph node chain irradiation in stage I–III breast cancer (joint study of the EORTC Radiotherapy Cooperative Group and the EORTC Breast Cancer Cooperative Group). EORTC Study No. 10925/22922A survey of the Breast International Group (BIG) to assess the attitude of patients aged <35 years, with early breast cancer, toward the risk of loss of fertility related to adjuvant therapies. BIG 3-98/EORTC 10002LAMANOMA: Conservative local treatment versus mastectomy after induction chemotherapy in locally advanced breast cancer: a randomized phase III study. BIG 2-00/EORTC Study No. 10974/22002p53 study: First prospective intergroup translational research trial assessing the potential predictive value of p53 using a functional assay in yeast in patients with locally advanced/inflammatory or large operable breast cancer, prospectively randomized to a taxane versus non-taxane regimen. BIG 1-00/EORTC 10994After mapping of the axilla: radiotherapy or surgery AMAROS. EORTC 10981/22023A randomized phase II–III trial evaluating the efficacy of capecitabine and vinorelbine in anthracycline and taxane pretreated metastatic breast cancer. EORTC 10001/160010Phase I study of lonafarnib (SCH 66336) in combination with Herceptin plus paclitaxel in HER-2 neu overexpressing breast cancer. EORTC 10051/16023MINDACT trial: A prospective, randomized study comparing the Amsterdam 70-gene expression signature (Mammaprint) with common clinical pathological criteria in selecting patients for adjuvant chemotherapy in node-negative breast cancer. BIG 3-04/EORTC 10041

Molecular Profiling of Bladder Tumors Based on the Detection of FGFR3 and TP53 Mutations

On a routine basis we performed systematic molecular screening for FGFR3 and TP53 mutations in 121 bladder tumors. We then specifically analyzed the predictive value of the recurrence of FGFR3 and TP53 genotypes in superficial lesions. The FGFR3 gene was analyzed by direct sequencing of exons 7, 10 and 15, whereas TP53 status was determined using the p53 functional assay in yeast. We identified a missense FGFR3 mutation in 66% of pTa, 26% of pT1 and 12% of pT2 tumors. Of activating FGFR3 mutations 54% and 85% were found in low G1 and intermediate G2 grade tumors, respectively, but in only 20% of high grade G3 tumors. We detected inactivating TP53 mutations in 10% of pTa, 42% of pT1 and 58% of pT2 tumors. Moreover, TP53 mutations were found only in 23% of grade G1 and 3% of grade G2 tumors but in 44% of high grade G3 tumors. When the 2 genotypes were combined, we observed that 58% of pTa tumors had the (mutant FGFR3, WT TP53) genotype, whereas 58% of invasive lesions harbored the inverse genotype (WT FGFR3, mutant TP53). The (mutant FGFR3, WT TP53) genotype and the (WT FGFR3, mutant TP53) genotype were detected in 23% and 38% of pT1G3 tumors, respectively. In the subgroup of 92 patients with superficial pTa-T1 bladder tumors we did not find that the TP53 or FGFR3 genotype alone or combined had a predictive value for tumor recurrence. Our data again represent solid proof for the pivotal role of FGFR3 and TP53 mutations in superficial and invasive bladder tumors, respectively. However, other molecular markers should be identified for borderline pT1G3 bladder tumors, which are probably at the crossroads of these 2 distinct molecular pathways.

Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-2'-deoxyguanosine DNA adducts: relevance for gastrointestinal cancer

Nitrosated glycine derivatives react with DNA to form O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) adducts concurrently. O6-CMdG is not repaired by O6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O6-CMdG and O6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with >50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (>80%) GC-->AT transitions, KDA produced almost equal amounts of transitions (GC-->AT) and transversions (GC-->TA and AT-->TA). This difference is probably due to a different mode of base mispairing for O6-CMdG compared with O6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer.

Predicting the transactivation activity of p53 missense mutants using a four-body potential score derived from Delaunay tessellations

We describe a novel statistical scoring method based on a computational geometry approach to predict the functional impact (transactivation activity) of missense mutations in the DNA-binding domain (DBD) of the tumor suppressor TP53, which is the most frequently mutated gene in human cancer. Residual scores (RS) for each residue were calculated to reflect differences in the compositional preferences of four nearest-neighbor residues between mutant and wild-type proteins. The RS were then combined into a residual score profile (RSP) representing the RS values for all 194 residues in the DBD. Mutants were grouped into functional categories based on their transactivation activities experimentally measured in yeast functional assays using p53-response elements from eight different promoters. While these functional categories showed significant differences in average RS, the latter lacked resolution power to predict the transactivation activities of individual mutants. In contrast, using decision tree models, we found that the RSP predicted transactivation with an accuracy varying between 64.2% and 78.5% depending on the promoter. Lastly, we used the best model to predict the functional outcome of all missense mutants in the DBD of p53 and compared the predictions with their frequency of occurrence in human cancers. We found that mutants predicted as functional (F) accounted for approximately 14% of all missense mutants found in cancers, while mutants predicted as nonfunctional (NF) represented approximately 86% of the mutants. These results show that this computational approach provides a fast and reliable method for predicting the functional impact of p53 mutants associated with cancer.

Analysis of p53 status in human cell lines using a functional assay in yeast: Detection of new non-sense p53 mutation in codon 124

p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.

P53 pathway dysfunction in primary childhood ependymomas

Childhood ependymoma remains a major therapeutic challenge despite surgery, chemotherapy, and irradiation. We hypothesized that p53 function might be abrogated in ependymomas and implicated in their resistance to anti-cancer therapy. Primary ependymomas at diagnosis or relapse from 24 children were analyzed for p53 pathway, using a functional assay in yeast, RT-PCR, Western blot analysis, and/or immunohistochemistry for TP53 mutation, p14(ARF) deletion and promoter hypermethylation, MDM2 and PAX5 expression, respectively. p53-mediated response to radiation-induced DNA damage was evaluated using Western blot and flow cytometry analysis in two ependymoma xenograft models, IGREP37 and IGREP83, derived from primary anaplastic childhood ependymomas. No TP53, MDM2, p14(ARF), PAX5 gene abnormalities were detected in the primary ependymomas tumors and xenografts tested. Interestingly, despite the lack of these abnormalities, p53 induced p21-mediated G(1) growth arrest in response to irradiation was altered in the IGREP37 xenograft tumors. Although irradiation induced necrosis and apoptotic cell death, IGREP37 tumors were moderately sensitive to radiation therapy in vivo. In contrast, irradiation yielded significant tumor growth delays and tumor regressions in the p53 functional IGREP83 xenografts. Alterations in p53-mediated growth arrest in ependymomas might be implicated in the radio-resistance of these tumors and demand further evaluation.

OsWRKY03, a rice transcriptional activator that functions in defense signaling pathway upstream of OsNPR1

WRKY family proteins are a class of plant specific transcription factors that involve in many stress response pathways. It has been shown that one Arabidopsis WRKY protein, AtWRKY29/22, is activated by MAP kinase signaling cascade and confers resistance to both bacterial and fungal pathogens. However, little is known about the biological roles of WRKY proteins in rice. In this study, we investigated the expression patterns of rice AtWRKY29/22 homolog, OsWRKY03, under different conditions, and also its possible role involved in plant defense. Our results showed that OsWRKY03 was up-regulated by several defense signaling molecules or different treatments. Further analysis revealed that the expression of OsWRKY03 was light dependent. Transcriptional activation activity of OsWRKY03 was also demonstrated by yeast functional assay. Transient expression of OsWRKY03-GFP fusion protein in onion epidermis cells showed that OsWRKY03 was a nuclear localized protein. OsNPR1 as well as several other pathogenesis-related genes, such as OsPR1b, phenylalanine ammonia-lyase (ZB8) and peroxidase (POX22.3), were induced in OsWRKY03-overexpressing transgenic plants. These results indicated that OsWRKY03 is located upstream of OsNPR1 as a transcriptional activator in salicylic acid (SA)-dependent or jasmonic acid (JA)-dependent defense signaling cascades.

Quantitation of p53 nuclear relocation in response to stress using a yeast functional assay: effects of irradiation and modulation by heavy metal ions

Many regulatory proteins undergo transient nuclear relocation under physical or chemical stress. This phenomenon is, however, difficult to assess due to the lack of sensitive and standardized biological assays. Here, we describe a new quantitative nuclear relocation assay (QNR), based on expression in yeasts of chimeric proteins in which an artificial transcription factor is fused to a target protein acting as driver for relocation. This assay combines the experimental versatility of yeast with quantitation of nuclear relocation at low levels of protein expression. We have assessed the nuclear relocation of yeast Yap1 and human p53, two transcription factors that relocate to the nucleus in response to oxidative-stress and DNA damage, respectively. We show that p53 efficiently drives the relocation of the chimeric reporter in response to irradiation and that this process requires the C-terminal nuclear export signal (NES). Cd2+ and Hg2+, two metal ions inducing DNA damage as well as conformational changes in p53, have opposite effects on p53 relocation in response to DNA damage. Whereas Hg2+ effects are synergistic to DNA damage, Cd2+ inhibits relocation and sequesters p53 into the cytoplasm. These results demonstrate the effectiveness of QNR to investigate the regulation of p53 shuttling in response to stress signals including suspected environmental carcinogens.

Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16(INK4a) promoter methylation was estimated using comparative methylation-specific real-time PCR. p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16(INK4a) promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.

The Last Reaction Producing Brassinolide Is Catalyzed by Cytochrome P-450s, CYP85A3 in Tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.

Functional analysis of p53 gene and the prognostic impact of dominant‐negative p53 mutation in endometrial cancer

In addition to the loss of function, mutant p53 can possess a dominant-negative effect on wild-type p53 and may also exert gain-of-function activity. It is not clear whether the functional status of p53 mutation contributes to differences in outcome in endometrial cancer. We collected a total of 92 RNA samples of high quality from endometrial cancer tissues, and the samples were subjected to yeast functional assay and sequencing for p53 mutations. The detected mutant p53 genes were further investigated for their dominant-negative activity using a yeast-based transdominance assay. p53 mutation was found in 24 out of 92 (26.1%) tumors, of which 10 exhibited no dominant-negative activity (recessive mutation) and 14 showed dominant-negative activity. Dominant-negative p53 mutation was related to advanced stages (p = 0.01), nonendometrioid type tumors (p = 0.01) and grade 3 tumors (p = 0.04). The patients with dominant-negative mutation had significantly shorter survival than patients with no mutation (p < 0.0001) and those with a recessive mutation (p = 0.01) in the p53 gene. No difference in survival was found between the patients with tumors harboring a recessive p53 mutation and those with tumors harboring a wild-type p53. Multivariate analysis revealed that dominant-negative p53 mutation (p = 0.019), FIGO stage (p = 0.0037) and histologic subtype (p = 0.014) were independently related to patient survival. Dominant-negative p53 mutation was the most important prognostic factor for stage III/IV endometrial cancer (p = 0.0023). In conclusion, dominant-negative p53 mutation is often found in advanced stages and aggressive histologic subtypes of endometrial cancer and it is a strong predictor of survival of patients with advanced endometrial cancer. To elucidate further the role of p53 mutation in endometrial cancer, it is necessary to investigate gain-of-function activity involving dominant-negative p53 mutant proteins.

Functional analysis of chemically-induced mutations at the flounder TP53 locus, the FACIM assay

A functional assay was developed in yeast to identify mutations induced by DNA-damaging agents at the flounder TP53 locus. This assay named FACIM for functional analysis of chemically-induced p53 mutations, is based on the assumption that most genotoxin-induced mutations inactivate transcriptional activity of the TP53 protein. The functional status of the protein expressed in yeast was measured using a p53-responsive reporter gene. The FACIM assay was used to evaluate the mutagenesis of the flounder TP53 exposed in vitro to benzo[ a]pyrene diol epoxide (BPDE). A dose-dependent increase of p53 mutation rate was observed with increasing concentrations of BPDE and extension of exposure time. Flounder TP53 gene appeared highly sensitive to point mutations since most of those identified targeted different nucleotides. Mutated base-pairs corresponded predominantly to guanines located on the non-transcribed strand of the DNA. The general distribution of mutations along the flounder TP53 protein was different from that identified in the human homologue suggesting species-differences in mutagenesis of the TP53 gene. Most of flounder TP53 mutants were defective for transactivation and cell growth regulation but some maintained a partial wild-type phenotype. This functional assay in yeast could be used for both evaluation of the genotoxic potency of chemicals or environmental samples and screening of p53 mutations in fish tumours.

Clinical implications of p53 mutation analysis in bladder cancer tissue and urine sediment by functional assay in yeast.

In the present study we correlate the p53 gene mutations in tumour tissue with urine sediment using a functional assay in yeast, and relate the p53 status to the outcome in a group of patients with transitional cell carcinoma of the bladder. The p53 mutations were found in three of 30 (10%) Ta/T1 tumour tissue samples and in two of 20 (10%) corresponding urine sediments. In the stage T2-T4 tumour p53 mutations were found in tumour tissues and urine sediments in 13 of 31 (42%) and in seven of 18 (39%) samples, respectively. In 80% (8/10) of cases, the p53 mutations found in tumour tissue were re-detected in urine sediment. Median follow-up was at 20 months. Disease recurred in 18 of the 61 patients (30%) with a median time of 5 months. In Ta/T1 tumours the frequency of recurrence was 37% (11/30) compared with 23% (7/31) of T2-T4 tumours. The 3-year overall survival (OS) was 82% (50/61). The p53 status was significantly associated with stage (P = 0.0077, two-sided Fisher's exact test), grade (P < 0.001) and lymph node involvement (P = 0.027). There was an association between the p53 mutations and shorter OS (P = 0.033; log-rank test); however in a multivariate analysis adjusted for stage, grade, lymph node status and age the p53 mutation was not an independent predictor of survival. There was no correlation of the p53 status with decreased disease-free survival (P = 0.8; log-rank test). The data presented indicate that the yeast functional assay is a useful method for p53 gene mutation analysis in tumour tissue and p53 mutation can be re-detected in urine sediment, but further validation of the assay in non-invasive screening for p53 mutations is needed.

Spectrum of p53 mutations in biopsies from breast cancer patients selected for preoperative chemotherapy analysed by the functional yeast assay to predict therapeutic response

The most frequent alteration detected in breast cancer cells is an inactivation of the tumor protein p53. Numerous studies revealed that p53 mutations are an independent prognostic indicator of a shorter period of overall and disease-free survival. Meta-analysis of these investigations clearly showed that prognostic significance cannot be achieved by indirect assessment of the p53 status by immunohistochemistry. Therefore, the tumor RNA-based functional p53 assay in yeast (functional assay of separated alleles in yeast, FASAY) appears to be an attractive option to generate clinically relevant information without the need of microdissection. We describe FASAY analyses of 50 biopsies taken before pre-operative anthracycline/taxane chemotherapy to evaluate the predictive value of p53 mutations for this common combination of substances. Wild-type p53, present in 22 samples, resulted in numerous white colonies with a low background of red colonies that was consistently below 8%. Biopsy samples containing mutated alleles gave rise to many red colonies accompanied with variable numbers of white colonies. With one single exception, all biopsies containing mutated p53 resulted in more than 8% red colonies. In 25 samples (53%), 23 single and 2 double mutations of the p53 gene were confirmed by sequencing of DNA from the yeast colonies. Six of the observed sequence insertions or deletions and 2 of the point mutations have not been reported previously. In accordance with an abolished or altered function as a transcriptional activator, most mutations affected the p53 DNA-binding domain, and one the tetramerization domain. Our results confirmed that the FASAY is sufficiently reliable to detect p53 mutations in breast tumor biopsies. In this pilot study with a limited number of patients to evaluate the predictive value of p53 mutations for an anthracycline/taxane combination therapy in the neoadjuvant setting, stable disease was observed more often in patients with wild-type p53 but statistical significance was not quite reached for this clear trend.

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.

Clinical evaluation of p53 mutations in urothelial carcinoma by IHC and FASAY

Objectives To assess the clinical benefit of the methods of detection of p53 mutations in human urothelial carcinoma. Methods A total of 75 surgical specimens of urothelial carcinoma were analyzed using a yeast functional assay (FASAY) and immunohistochemistry (IHC), in combination with sequencing analysis. Results Of the 75 specimens, 24 (32.0%) were positive (mutant) by FASAY and 23 (30.7%) were positive by IHC. The sequencing analysis confirmed that all 24 FASAY-positive tumors harbored mutations, and no mutations were detected in any FASAY-negative tumors. In contrast, nuclear accumulation of p53 protein was detected in 9 (17.6%) of 51 tumors with no mutation, and 10 (41.7%) of 24 tumors with mutation showed no positive staining on IHC. The mutations detected by FASAY and IHC were both associated with stage and grade, but null mutations of p53 were not associated with stage. Concerning chemosensitivity, 6 (85.7%) of 7 responders harbored p53 missense mutations in at least one allele ( P = 0.01), and only 4 (57.1%) were judged positive by IHC ( P = 0.13). Conclusions FASAY is more accurate than IHC in detecting the various types of p53 mutations, suggesting that a comprehensive approach for the detection of p53 mutations may be essential to elucidate their clinical significance.

1078: Use of a Yeast Functional Assay to Determine the Frequency of P53 Alterations in Localized Prostate Cancer

1078: Use of a Yeast Functional Assay to Determine the Frequency of P53 Alterations in Localized Prostate Cancer

Loss of p53 transcriptional activity in hepatocellular carcinoma evaluated by yeast-based functional assay: comparison with p53 immunohistochemistry

We studied the transcriptional activity of p53 protein in 50 tissues of hepatocellular carcinoma (HCC) using a yeast functional assay. In this assay, red yeast colonies indicate that p53 protein cannot bind to its specific domain and has lost its transcriptional activity. We also clarified whether mutant p53 protein could inactivate wild-type p53 protein in a transdominant manner using a modified yeast assay. In addition, we examined whether immunohistochemically detectable p53 protein was functionally inactive. The incidence of p53 inactivation was significantly higher in tumors with capsular invasion. Out of 21 tumors diagnosed with p53 mutations, 11 exhibited >75% red colonies, and all contained missense mutations. In these tumors, p53 function was lost because there was supposedly no intact p53 gene on either allele. One missense mutant produced <60% red colonies, but it was also considered inactive as a p53 protein heterotetramer because of its transdominant activity. In 7 of the remaining 9 tumors, p53 was considered to be mutated on one allele and intact on the other. All of these 7 tumors contained nonsense or frameshift mutations and had no transdominant activity, which suggested that p53 function remained intact. Alternately, immunohistochemical analysis demonstrated that all of the tumors with missense mutations were positively immunostained, whereas those that contained nonsense or frameshift mutations were negatively stained. Consequently, positively immunostaining tumors mostly coincided with p53-inactive tumors. These yeast-based assays suggested that p53 function was retained in some mutant cases. Immunohistochemistry was helpful in screening functionally inactive p53 protein in HCCs.

The European flounder (Platichthys flesus) TP53 functions as a temperature-sensitive transcription factor which inhibits cell growth in yeast.

Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates. In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein. We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE). This transcriptional activity was completely abrogated in the Val147Glu TP53 mutant previously identified in two flounder hepatic hyperplasia. In addition, we showed that the wild-type (wt) flounder TP53 but not the Val147Glu mutant inhibits cell growth when expressed in yeast. We finally reported that transcription regulation and growth inhibition by the wild-type flounder TP53 is temperature-dependent. The flounder TP53 optimal temperature appeared lower than those reported for the Xenopus and human homologues.